The Dark Art of Blood Cultures

Affiliations: 1: bioMérieux, Inc., Durham, North Carolina;
2: Washington University School of Medicine in St. Louis, St. Louis, Missouri;
3: Duke University School of Medicine, Durham, North Carolina;
4: Washington University School of Medicine in St. Louis, St. Louis, Missouri

In the clinical microbiology laboratory, blood is a critical diagnostic sample that, in the majority of cases is sterile (or is it?). However, when microbes gain access to and multiply in the bloodstream, it can result in life-threatening illness including sepsis. Mortality rates from bloodstream infection and sepsis range from 25% to 80%, killing millions of people annually. Blood cultures are a vital technology used in the microbiology laboratory to isolate and identify microbes and predict their response to antimicrobial therapy.

The Dark Art of Blood Cultures, edited by Wm. Michael Dunne, Jr., and Carey-Ann D. Burnham, surveys the entire field of blood culture technology, providing valuable information about every phase of the process, from drawing samples to culture methods to processing positive cultures. The Dark Art of Blood Cultures is organized around several major topics.

History of blood culture methods. Details the timeline of blood culture methods from manual through automated and describes the technological development of the leading automated blood culture systems (Bactec, BacT/Alert, and VersaTREK).

Detection of pathogens directly from blood specimens. Describes currently available CE marked and FDA-cleared commercial tests using both phenotypic and genotypic markers, including their strengths and limitations.

The workflow of culturing blood. Includes best practices from specimen collection to culture system verification, processing positive cultures for microbe identification and antibiotic susceptibility determination, along with the epidemiology of positive blood cultures and the value of postmortem blood cultures.

Microorganisms in the blood. Examines the concept of a blood microbiome in healthy and diseased individuals.

The Dark Art of Blood Cultures is a resource that clinicians, laboratorians, lab directors, and hospital administrators will find engaging and extremely useful.

From an historic point of view, one can divide the evolution of blood culture technology into four distinct “archeological” periods that I refer to (tongue in cheek) as the “Manualithic” (pre-1970), the “Bactecene” (1970 to 1990), the “Continuous Monitorassic” (1990 to 2000), and the “Ampliaissance” (post-2000) ages (Fig. 1). Each of these denotes a quantum shift in the use of technology to increase the sensitivity and decrease the time-to-detection of microorganisms in blood. While the rate of improvement in both speed and sensitivity has clearly reached steady state over recent years, and the ultimate goal of directly detecting microorganisms causing bacteremia/fungemia/sepsis from blood has not yet been achieved, progress is most definitely moving forward and the tools being developed for that purpose are impressive. However, before we find out how far we have come, it is best to review where the concept of finding microorganisms in blood began, and some historians would conclude that it started with Athanasius Kircher (Fig. 2).

Chapter 1 took us on a time-travel journey spanning the evolution of blood cultures. All the experimentation and discovery since the 17th century have led to a common understanding of which microorganisms cause bloodstream infections and what essential nutrition is required to grow and recover them from blood. Even today, blood culture remains the best option to determine microbial causes of sepsis. Detection of microbial nucleic acids in blood by PCR or sequencing may not represent the presence of viable organisms (see chapter 13 on the blood microbiome). Often, manual culture methods are used as a reference standard for newer iterative protocols like current-generation automated blood culture systems (1). While the routine use of manual blood culture methods in modern diagnostic microbiology laboratories in 2017 is no longer commonplace, the practice remains quite prevalent in the developing world where purchase and maintenance of automated blood culture instruments are both unaffordable and impractical. But in the developed world, manual blood culture protocols often serve as a backup plan if automated systems experience problems. The comedian Mitch Hedberg once said, “An escalator can never break: it can only become stairs.” The same holds true for automated blood culture systems. Regardless of how blood cultures are performed, however, the same set of parameters must be attained to ensure optimal microbial recovery. The list of best practice benchmarks includes the following and can also be found in chapter 10:

In 1917, Mildred Clough first demonstrated the usefulness of lysis-centrifugation blood culture when she used it to isolate Mycobacterium tuberculosis from blood (1, 2). Using this processing method, she was able to recover colonies of M. tuberculosis on solid agar media in just two weeks. The process was complex and time-consuming, and, despite the desirable results, lysis-centrifugation blood culture was largely abandoned until the mid-1970s.

In the mid-1950s Dr. William Johnston and colleagues from Purdue University founded a company that specialized in developing instruments to detect very low levels of carbon-14, which were marketed for use in air quality testing. In the late 1950s, the company was sold to an investor who took it in the direction of seeking government contracts to study beta radiation and develop sensitive detection devices. The desire to transition the business to development of commercial products for the private sector led to a collaboration with scientists at Johns Hopkins University School of Medicine and the development of the Bactec 110 (Fig. 1A), the company’s prototype bacterial detection system. The instrument accommodated 10 sample bottles on a rotatory device and utilized a dual-needle adaptor to penetrate the rubber septum and sample headspace gas from bottles. The technology was based on Johnston’s tritium air monitor, detecting carbon-14-labeled CO2 produced by bacteria from radiolabeled substrates in the medium (1, 2).

This chapter introduces the BacT/Alert, the first automated blood culture system, and its descendants. The first part of the chapter covers the historical context in which the system was developed and some of the key design considerations that were intended to close gaps in blood culture practice in the early 1990s. The second part of the chapter outlines the evolution of the BacT/Alert culture media, their performance, and design changes to subsequent instruments in the BacT/Alert family tree.

Over the past three-plus decades, Difco Laboratories (Detroit, MI) and then TREK Diagnostic Systems (Thermo Scientific Microbiology, Waltham, MA) have developed several iterations of continuous-monitoring blood culture systems (CMBCSs). Similar to other CMBCSs, these systems allow for incubation of inoculated blood culture bottles with simultaneous monitoring of positivity by indirect measurement of bacterial growth. However, the unique approach toward monitoring growth distinguishes the TREK systems from other CMBCSs.

Sepsis is a global problem and laboratory methods must be optimized to effect rapid, positive patient outcomes. The proverbial reference standard method for diagnosis, the actual culture of blood, suffers from a variety of preanalytical issues such as sufficient blood volume, prior antimicrobial treatment, time from sampling to incubation, and analytical issues of prolonged turnaround time until final identification and availability of antimicrobial susceptibility testing results. Advancements have occurred in molecular methods to provide rapid microorganism identification and resistance gene detection (e.g., mecA in Staphylococcus spp.) from positive blood culture broths. In a meta-analysis of these new methods, a significant reduction in time to targeted treatment was observed (1). However, these methods are dependent on having a positive blood culture, which is subject to the preanalytical issues described above. The ability to directly identify pathogens from blood specimens would greatly reduce the time to identification and time to optimization of therapy, as well as potentially contributing to avoiding unnecessary antimicrobials and/or discontinuation of treatment in patients with negative testing results. Direct detection methods for viruses from patient specimens have benefited from advances in molecular methods, which is largely because of the high viral loads present in those specimens. In contrast, the majority of septic patients have a paucity of bacteria or fungi per milliliter of blood, necessitating the need for larger blood volumes to increase blood culture sensitivity (2–4).

In pediatrics as in adult medicine, the clinical microbiology laboratory plays an integral role in providing accurate and timely data to aid in the diagnosis, treatment, and monitoring of various infectious diseases. Because of the associated morbidity and mortality with bloodstream infection (BSI), blood cultures are among the most critical diagnostic tests in pediatric laboratory medicine. Despite the critical nature of the pediatric blood culture, there remains a great deal of myth and misunderstanding surrounding these cultures. The unique challenges associated with pediatric blood cultures include the wide range of patient blood volumes making one standard recommended culture volume impossible, an ever evolving epidemiology of sepsis due in large part to the availability of various vaccines, and a diverse range of clinical presentations due to the dynamic patient population, ranging from neonates through the late teenage years. This chapter focuses on these uniquely pediatric challenges.

The detection of microorganisms in the setting of bloodstream infection is one of the most important functions of the clinical microbiology laboratory. A number of advances and practice changes in health care, including hematopoietic stem cell transplantation (HSCT) and solid organ transplantation (SOT) and resulting immunosuppressive regimens, as well as the aging population, emergence of antimicrobial resistance, and increased use of invasive procedures in health care settings (as well as improvements in blood culture methods), have resulted in changing epidemiology of bloodstream infections over the past 4 decades. This chapter will discuss trends in the epidemiology of positive blood cultures.

Rapid and accurate detection of sepsis is critical, yet errors in collection lead to unnecessary testing, use of antimicrobial agents, and prolonged hospitalizations. Strict attention to the various components that compose the blood culture collection process can help to reduce factors leading to rising costs in health care as well as inaccurate test results.

It can be said that the diagnosis of bloodstream infections is one of the most important roles of the clinical microbiology laboratory; the mortality rate associated with bloodstream infection ranges from 25 to 80% (1, 2). There are a number of factors that contribute to the mortality rate subsequent to bloodstream infection, with time to appropriate antimicrobial therapy frequently cited as one of the most important variables correlating with clinical outcome (3–5). Herein, we describe procedures for optimization of positive blood culture specimens and describe current and future methodologies to augment blood culture to diagnose bloodstream infection.

Early and appropriate antimicrobial therapy of sepsis is associated with improved clinical outcomes, and the laboratory identification of the etiological agent of fungemia and/or mycobacteremia is very important for successful outcome. Fungi and some mycobacteria grow more slowly than many common pathogenic bacteria, and specialized broth culture media and methodologies are available for their isolation. Not surprisingly, there are reports of fungi, primarily yeast, being recovered more effectively using mycobacterial blood culture media compared with routine blood culture bottles intended for the detection of aerobic and anaerobic bacteria (1, 2). The laboratory diagnosis of mycobacteremia and fungemia often requires special consideration with the selection of blood culture testing and interpretation of results.

Many surfaces on the human body are continually exposed to microorganisms in the external environment and become colonized before or shortly after birth with a normal resident microbiota, the collection of microorganisms in a particular environment, which matures over time and persists throughout life. These nonsterile surfaces include the skin, mucous membranes, lower gastrointestinal tract, upper respiratory system, and anterior urethra. The community of microorganisms harbored in or on these sites is a highly complex mixture that varies in composition depending on nutrient availability, moisture, temperature, pH, and other environmental conditions. The composition of the normal microbiota can also be affected by host factors such as age, nutrition, and immune status.

Autopsy has historically played a critical role in our understanding of normal anatomy, disease and treatment efficacies, or adverse effects of medications. Despite the importance of autopsy as a teaching tool, it is now limited to an ancillary role in modern medical education (1). This effect may continue to be amplified as physicians who did not observe autopsies during their training are less likely to request an autopsy later on in the course of their careers (2). In the 1970s, the Joint Commission on Accreditation of Healthcare Organizations (JCAHO) eliminated the requirement for a minimum autopsy rate from its accreditation process, and Medicare stopped reimbursing for autopsies in 1986 (3). Reasons physicians do not request an autopsy have reportedly included trepidation regarding the lack of training on how to seek autopsy permission, fear of offending the family, and fear of malpractice litigation. In addition, confidence in contemporary diagnostic technology and the ever present desire to reduce health care spending have also been cited as reasons (2, 4–9). Given the forces aligned against this procedure, autopsy rates have declined in the United States and other Western countries over the past 3 decades (4). Unfortunately, the sequelae of this decline include the loss of an established teaching tool along with the inability to identify and correct clinical errors and missed diagnoses (4).